Competition based-detection assays

ABSTRACT

Disclosed herein are methods and kits which are useful for detecting presence of an enzyme and the relative amount of glycan associated with the enzyme in a test sample based upon the enzyme&#39;s ability to competitively inhibit the binding of a ligand in such test sample. The present invention provides the ability to evaluate cell culture conditions and optimize the desired glycoform content of recombinantly prepared enzymes.

RELATED APPLICATION(S)

This application claims the benefit of U.S. Provisional Application No.61/494,643, filed Jun. 8, 2011 (Attorney Docket No. SHIR-024-001). Theentire teachings of the above application are incorporated herein byreference.

BACKGROUND OF THE INVENTION

Glycoproteins and glycoenzymes are proteins that contain apost-translational modification wherein oligosaccharide chains (known asglycans) are covalently attached to the protein's or enzyme'spolypeptide side chains. This process, which is known as glycosylation,is one of the most abundant protein post-translational modifications. Itis estimated that more than half of all cellular and secretory proteinsare glycosylated. (Apweiler et al., 1999, Biochim. Biophys. Acta 1473:4-8). Although mammalian glycoprotein oligosaccharides, for example, areconstructed from a limited number of monosaccharides, their structuraldiversity is vast due to complex branching patterns. Glycoproteins,therefore, represent a diverse group of modifications, and variants ofglycoproteins or glycoenzymes (which are known as glycoforms) can impactprotein or enzyme activity or function. The ability to evaluate anddistinguish specific glycan structures during the preparation ofrecombinant enzymes can accordingly provide valuable informationrelating to recombinant enzyme development and further optimization ofthe desired glycoform content of such recombinant enzymes.

Conventional techniques which are routinely employed for glycoproteinand glycoenzyme analysis include mass spectrometry, lectin affinitychromatography and western blotting. Although these conventional methodsof analysis are generally accurate, they are time consuming, requirepurification of the protein, and some, such as mass spectrometry,require specific expertise and are technically challenging. (Wang etal., 2006, Glycobiol. Epub.; Qiu et al., 2005, Anal. Chem. 77:2802-2809;Qiu et al., 2005, Anal Chem. 77:7225-7231; Novotny et al., 2005, J. Sep.Sci. 28:1956-1968). Accordingly, these issues make the routine use ofsuch technologies impractical for high-throughput monitoring of enzymeglycosylation, especially during process development and manufacturing.Such technologies may also present challenges to a typical researchlaboratory attempting to study the impact of glycosylation on thebiological properties of proteins and enzymes.

Traditionally, to provide a quantitative assessment of the glycanstructure of a glycoprotein, lectin array platforms required the use ofeither a reliable glycoprotein-specific antibody or direct conjugationof a fluorescent dye to the glycoprotein. These antibody-based detectionstrategies are limited by the fact that antibody recognition of a givenglycoprotein or glycoenzyme may be blocked or reduced depending on thetype of glycan structure linked to the protein or enzyme, therebyallowing recognition of only a subset of the total glycoprotein pool andnot the range of potential glycan structures. Antibody-based recognitionmay also require multiple binding and wash steps, which can add time andcomplexity to an analysis. While these problems can be circumventedusing direct labeling of the glycoprotein, direct labeling remainslimited to pure preparations of material, since the labeling techniquesdo not discriminate among proteins. Accordingly, direct labeling cannotbe used for “dirty” or in-process samples. The utility of currentlyavailable methods for glycan analysis may be further limited becauselarge quantities of highly purified materials may not readily beavailable from in-process test samples. Furthermore, purified materialmay only represent a subset of the initial glycoform population becausethe purification process is typically selective for certain glycanstructures.

The identification and characterization of protein and enzyme glycoformsis essential in the development of recombinant proteins and enzymes. Forexample, glycosylation of recombinantly-prepared enzymes must frequentlybe controlled during production to maintain the efficacy and safety ofsuch recombinant enzymes, and cell culture conditions can affect thecarbohydrate structures of glycoproteins. Further understanding of cellculture conditions that can impact the carbohydrate structures ofrecombinantly-prepared proteins or enzymes is also important for thedevelopment of an effective and robust recombinant production process.

Improved methods and compositions are needed for the rapid, direct andsystematic identification and evaluation of the glycan structures of agiven protein or enzyme and their variant glycoforms. High throughputmethods and compositions that are capable of efficiently assessing anddistinguishing among a diverse range of glycosylation states orglycoforms, as well as determining the relative differences in theamount of glycans associated with such glycosylation states orglycoforms, would provide valuable information for drug discovery anddisease therapeutics, provide valuable tools regarding ongoing research,and facilitate the optimization of recombinant production processes.

SUMMARY OF THE INVENTION

The present invention provides novel methods, assays and compositionsfor the accurate and rapid identification and/or detection of variousglycoforms of enzymes and the relative amount of glycan associated withsuch glycoforms. In particular, the present invention relies upon theability of an enzyme of interest to competitively inhibit the binding ofa ligand to detect such enzyme's presence in a test sample, as well asto determine the relative amount of glycan associated with such enzyme.The methods, assays and compositions disclosed herein also provide novelstrategies for analyzing the different glycoforms of unpurified proteinsor enzymes in cell culture harvest test samples. The methods, assays andcompositions disclosed herein also provide novel strategies foranalyzing the relative amounts of glycan associated with such differentglycoforms. Furthermore, the present invention provides the ability todetect and distinguish among different glycoforms or glycovariants of anenzyme in upstream harvest test samples, thereby facilitating theoptimization of cell culture conditions that affect the viable glycoformcontent of recombinantly-prepared enzymes. Even further, the presentinvention provides the ability to detect and distinguish among therelative amount of glycan associated with various glycoforms orglycovariants of an enzyme in upstream harvest test samples, therebyfurther facilitating the optimization of cell culture conditions thataffect the viable glycoform content of recombinantly-prepared enzymes.The methods and kits of the present invention are advantageously capableof determining the presence of glycosylated enzymes in a test sample, aswell as determining the relative amount of glycan associated with thoseglycosylated enzymes, irrespective of the presence of additionalcellular proteins, biological materials or other contaminants which maybe present in that test sample.

Disclosed herein are methods for detecting the presence of an enzyme(e.g., a recombinantly prepared enzyme) and the relative amount ofglycan associated with the enzyme in a test sample, such methodscomprising contacting the test sample with at least one capture agent(e.g., a lectin) under conditions appropriate for binding ofglycosylated enzyme in the test sample to the capture agent, whereinupon binding of glycosylated enzyme to capture agent a complex is formedwhich is referred to herein as a “bound enzyme.” Some embodiments alsocontemplate separation of the test sample from the bound enzyme producedby the previous step (e.g., using routine means such as washing)followed by detection of the extent to which the bound enzyme inhibitsbinding of a ligand to the capture agent. The extent to which the boundenzyme inhibits binding of the ligand to the capture agent is indicativeof the relative amount of glycan associated with the enzyme in the testsample.

Also disclosed are methods for detecting the presence of an enzyme(e.g., a recombinantly prepared enzyme) and the relative amount ofglycan associated with the enzyme in a test sample, wherein such methodscomprise the steps of contacting a test sample with at least one captureagent (e.g., a lectin) under conditions appropriate for binding of theglycosylated enzyme, and thereby forming a bound enzyme whenglycosylated enzyme is present. The methods of the present inventionalso contemplate separating the bound enzyme from the test sample andcontacting the bound enzyme with at least one ligand for the captureagent. In accordance with the present invention, the extent to whichsuch bound enzyme competitively inhibits binding of the at least oneligand to the capture agent is indicative of the relative amount ofglycan associated with the glycosylated enzyme of interest in the testsample. Conversely, the absence of competitive inhibition is indicativeof the absence of the glycosylated enzyme of interest in the testsample. The methods disclosed herein provide the ability to optimize thedesired glycoform content of one or more recombinant enzymes duringrecombinant preparation.

In one embodiment, the methods of the present invention further comprisethe step of fixing a capture agent (e.g., one or more lectins) onto asolid support (e.g., a microtiter plate or one or more populations ofbeads). In one embodiment, such solid support may comprise or be coatedwith avidin, streptavidin or a metal chelator such as a nickel chelate.If such solid support comprises avidin or streptavidin, the use ofderivatized lectins (e.g., biotinylated lectins) are preferred. If suchsolid support comprises a nickel chelate, the use of six consecutivehistidine residues (6His) as an affinity tag is preferred. For example,a capture agent may be a fusion protein which includes one or morehistidine (HIS) residues (e.g., at least one, at least two, at leastthree, at least four, at least five, at least six, at least eight, atleast ten, at least twelve, at least twenty, at least twenty five ormore HIS residues) at either the N- or C-terminus as an affinity tag tofacilitate fixing of that capture agent (i.e., the fusion protein) to asolid support.

In one embodiment of the present invention the capture agent comprisesone or more lectins. The lectins contemplated by the methods, assays andkits of the present invention include, for example, concanavalin A,wheat germ agglutinin, Jacalin, lentil lectin, peanut lectin, lensculinaris agglutinin, Griffonia (Bandeiraea) simplicifolia lectin II,Aleuria aurantia lectin, hippeastrum hybrid lectin, sambucus nigralectin, maackia amurensis lectin II, ulex europaeus agglutinin I, lotustetragonolobus lectin, galanthus nivalis lectin, euonymus europaeuslectin, ricinus communis agglutinin I, and any combinations thereof.

In another embodiment of the present invention the capture agentcomprises a receptor, or a binding fragment thereof, known todemonstrate affinity for or otherwise hind to one or more particularglycoforms of an enzyme. For example, mannose-6-phosphate (M6P) bindsthe mannose-6-phophate receptor (M6PR), and in one embodiment arecombinant fusion protein comprising the M6PR or a binding domainthereof (e.g., M6PR domain 9) may serve as the capture agent. In oneembodiment, the recombinant fusion protein capture agent may alsocomprise one or more histidine residues (e.g., 6His) to facilitatepurification, capture and/or fixing of the capture agent to a solidsupport. In one embodiment of the present invention, the capture agentcomprises the fusion protein M6PR(D9)6His.

Also disclosed is a method of determining the intrinsic enzymaticactivity of the ligand bound to the capture agent by contacting suchligand with a substrate, for example, a substrate which has knownreactivity with the ligand. In accordance with the methods of thepresent invention, the presence of intrinsic enzymatic activity isindicative of the presence of ligand bound to the capture agent.Alternatively, the absence of intrinsic enzymatic activity may beindicative of the absence of such ligand bound to the capture agent inthe test sample.

In one embodiment, the methods, assays and kits of the present inventioncontemplate determining intrinsic enzymatic activity by contactingligand bound capture agent with a substrate which is known topredictably react with the ligand of interest. For example, if theligand is agalsidase alfa the selected substrate may be4-nitrophenyl-α-D-galactopyranoside, if the ligand isgalactocerebrosidase the selected substrate may be4-nitrophenyl-β-D-galactopyranoside, and if the ligand is aryl sulfataseA the selected substrate may be p-nitrocatechol sulfate. The presence orabsence of intrinsic enzymatic activity of the bound ligand may bedetermined by means which are known to those of ordinary skill in theart. In one embodiment a quantitative assessment of the conversion ofsubstrate to product may be indicative of intrinsic enzymatic activityof the ligand. For example, in one embodiment, following contacting anenzyme (e.g., ligand) with a substrate, a relative increase in theformation of a product, or the conversion of substrate to product, ineach case of about 5%, 10%, 20%, 30%, 40%, 50% or more, or preferablyabout 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100% or more,may be indicative of intrinsic enzymatic activity of the ligand.Substrates contemplated by the present invention include, for example,4-nitrophenyl-α-D-galactopyranoside, 4-nitrophenyl-β-D-galactopyranosideand para-nitrocatechol sulfate.

Also disclosed herein are kits which are useful for detecting thepresence of glycosylated enzymes (e.g., a recombinantly preparedglycosylated enzyme) and the relative amount of glycan associated withthe glycosylated enzymes in a test sample. Such kits comprise at leastone capture agent (e.g., a lectin) capable of binding a glycosylatedenzyme, and at least one ligand which is competitive with suchglycosylated enzyme for binding the capture agent. In one embodiment thekits of the present invention comprise a solid support (e.g., a multiplewell microtiter plate), onto which may be fixed a capture agent (e.g.,the lectin sambucus nigra agglutinin).

In one embodiment, the kits of the present invention comprise a captureagent which is known to bind or demonstrate affinity for the targetedglycoform of the enzyme of interest (e.g., the M6PR(D9)6His fusionprotein), and a ligand which is known to compete with such enzyme forbinding to the capture agent. In one embodiment, such kits may alsocomprise a means of separating or removing excess test sample from thesolid support, for example by washing, or other routine means availableto one of ordinary skill in the art.

Also contemplated are kits which are capable of identifying multipleglycosylated enzymes and multiple glycoforms of those enzymes in thesame test sample. For example, the kits of the present invention maycomprise multiple capture agents (e.g., lectins) fixed onto one or moresolid supports (e.g., populations of inert beads), and thus provide thecapability of binding to multiple glycoforms of one or more enzymes inthe same test sample. The kits of the present invention may alsocomprise one or more ligands (each of which compete with a particularenzyme whose presence is suspected in a test sample for binding to theone or more capture agents) to determine the extent to which suchenzymes competitively inhibit binding of the ligands to the captureagents. Preferably, the selected ligand is known to predictably bind to,or react with, the selected capture agent, and in particular, to competewith the enzyme of interest for binding the capture agent. For example,if the enzyme is idursulfase the selected ligand may be agalsidase alfa,if the enzyme is heparan N-sulfatase the selected ligand may beagalsidase alfa, if the enzyme is aryl sulfatase A the selected ligandmay be agalsidase alfa. Based upon the binding specificity or reactivityof the test sample with the ligand, one having ordinary skill in the artmay use routine means to assess the extent to which the enzymecompetitively inhibits the binding of the ligand to the capture agent(e.g., by detecting the presence or absence of intrinsic enzymaticactivity of the ligand bound to the capture agent, e.g., by contactingthe ligand bound to the capture agent with a substrate known to reactwith the ligand, e.g., by quantitatively determining the conversion ofsubstrate to product).

The above discussed and many other features and attendant advantages ofthe present invention will become better understood by reference to thefollowing detailed description of the invention when taken inconjunction with the accompanying examples.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 schematically illustrates one embodiment of the present inventionin which biotinylated lectins are bound to streptavidin-coated plates,to which are added test sample materials containing differentpreparations of a given glycosylated enzyme which are allowed to bind.Unbound test sample materials are then removed by a wash step, andspecific detection of the bound enzyme is performed by the addition ofthe appropriate substrate and assay conditions.

FIG. 2 illustrates binding of agalsidase alfa to immobilized wheat germagglutinin (WGA) and concanavalin A (ConA) as determined by measuringthe enzymatic activity of agalsidase alfa (Replagal®) based on thesubstrate 4-nitrophenyl-α-D-gatactopyranoside.

FIG. 3 illustrates binding of galactocerebrosidase (GalC) to immobilizedwheat germ agglutinin (WGA), concanavalin A (ConA), and Sambucus nigralectin (SNA) as determined by measuring enzymatic activity of GalC usingthe substrate 4-nitrophenyl-β-D-galactopyranoside.

FIG. 4 illustrates binding of galactocerebrosidase (GalC) treated withincreasing concentrations of sialidase to immobilized Sambucus nigralectin (SNA), as determined by measuring enzymatic activity of GalCusing the substrate 4-nitrophenyl-β-D-galactopyranoside.

FIG. 5 illustrates linkage-specific binding of purified aryl sulfatase A(ARSA) containing sialic acid in either α-2,6 or α-2,3 linkages toSambucus nigra lectin (SNA), as determined by measuring enzymaticactivity of ARSA using the substrate p-nitrocatechol sulfate.

FIG. 6 illustrates binding of galactocerebrosidase (GalC) cell culturefrom different harvest test samples to Sambucus nigra lectin (SNA), asdetermined by measuring enzymatic activity of GalC using the substrate4-nitrophenyl-β-D-galactopyranoside.

FIG. 7 schematically illustrates one embodiment of the present inventionin which the M6PR(D9)6His fusion protein is bound to a nickelchelate-coated 96-well plate, to which are added test samples containingdifferent preparations of a given glycosylated enzyme which are allowedto bind. Unbound test sample material is then removed by a wash step,and specific detection of the bound enzyme is performed by the additionof the appropriate substrate and assay conditions.

FIG. 8 illustrates detection differences in the amount of aryl sulfataseA (ARSA) associated M6P using ARSA lots with known amounts of M6P.

FIG. 9 illustrates detection differences in the relative amounts ofsialic acid associated with idursulfase, heparan N-sulfatase (HNS), andARSA using agalsidase alfa lots with known amounts of sialic acid.

FIG. 10 illustrates detection differences in the relative amounts of M6Passociated with idursulfase, IINS, and ARSA using agalsidase alfa lotswith known amounts of M6P.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety. In addition, the materials,methods, and examples are illustrative only and are not intended to belimiting.

Disclosed herein are high throughput methods, assays, kits andcompositions for screening complex test samples for the presence ofglycosylated enzymes of interest or for determining changes inglycosylation of such enzymes. Also disclosed herein are methods andkits which are capable of detecting the intrinsic activity of an enzymein a test sample as a means of determining the presence of that enzymein the test sample. For example, in one aspect the present inventionrelies upon the intrinsic enzymatic activity of an enzyme to detect itspresence in a test sample. This is in contrast to antibody-baseddetection schemes which can be negatively influenced by changes inglycosylation, for example by hindrance of antibody recognition of theenzyme. Also disclosed herein are methods and kits which are capable ofdetermining the relative differences in the amount of glycan associatedwith glycosylated enzymes present in complex test samples. For example,in another aspect the present invention relies upon the intrinsicability of an enzyme to competitively inhibit binding of a ligand to acapture agent to detect the extent to which the enzyme inhibits bindingof the ligand to the capture agent. Without wishing to be bound bytheory, it is believed that the relative amount of glycan associatedwith a glycosylated enzyme of interest correlates to the extent to whichthe glycosylated enzyme inhibits binding of the ligand to the captureagent. Specifically, the present invention relates to the discovery thatthe intrinsic ability of an enzyme to competitively inhibit binding of aligand with demonstrated affinity and specificity for binding to acapture agent is consistent with the relative amount of glycanassociated with the glycosylated enzyme. The invention provides theability to study cell culture conditions and optimize the desiredglycoform content of biological samples, and in particular ofrecombinantly prepared enzymes.

In the context of the present invention, the term “glycan” refers to thecarbohydrate portion of a glycoprotein or glycoenzyme. Generally,glycans tend to be oligosaccharides or polysaccharides. The terms“glycoform” and “glycovariant” refer to an isoform of a protein orenzyme with a unique primary, secondary, tertiary, and/or quaternarystructure based upon the number and/or structure of the glycans attachedto such protein or enzyme. It is often the case that a singleglycoprotein may have over a thousand different glycoforms orglycovariants, all of which are based on differences in the glycanportion or glycosylation pattern of the glycoprotein. The term“glycosylation” refers to the process or result of adding saccharides toproteins and thus forming “glycoproteins”. Glycosylation includes bothN-linked glycosylation to the amide nitrogen of asparagine side chains,and O-linked glycosylation to the hydroxy oxygen of serine and threonineside chains.

The term “test sample” is used in its broadest sense and means anypreparation, preferably obtained from biological media or materials,including biologically or recombinantly derived media which may contain,among other things, naturally occurring or recombinantly preparedpeptides, polypeptides or proteins, enzymes, lipid or carbohydratemolecules, or glycosylated proteins or enzymes, or other samplesobtained from a recombinant media, including any fractions thereof. Thetest samples contemplated by the present invention are preferablyobtained from in-process or “dirty” biological systems, for example,those obtained during the preparation of a recombinant enzyme.

As used herein, the term “solid support” refers to any material thatprovides a solid or semi-solid structure with or upon which anothermaterial can be attached or fixed. Such materials include smoothsupports (e.g., metal, glass, plastic, silicon, and ceramic surfaces) aswell as textured and porous materials. Such materials also include, butare not limited to, gels, rubbers, polymers, dendrimers and othernon-rigid materials. Solid supports need not be flat. Supports includeany type of shape including spherical shapes (e.g., beads) and mayinclude multiple well microtiter plates, and may optionally be coatedwith proteins, resins or other similar reagents, such as for example,avidin, streptavidin, metal ions or chelates (e.g., a nickel chelate).

The present invention contemplates the use of one or more capture agentsto facilitate capture, immobilization or fixing of the glycosylatedenzyme of interest (e.g., capture or fixing of one or more glycosylatedenzymes onto a solid support). As used herein, the phrase “captureagent” refers to a compound or a material which demonstrates affinityfor or is capable of conjugating or associating with one or morespecific saccharide or carbohydrate moieties. Preferably the selectedcapture agent demonstrates specific or discriminatory affinity for onesaccharide moiety, such that the capture agent will only bind oneparticular glycoform of a given enzyme. In a preferred embodiment of thepresent invention, the capture agent is selected based upon its specificaffinity to one or more glycan structures. When contacted with such aglycan structure or glycosylated enzyme in accordance with the presentinvention the capture agent will form a complex referred to herein as a“bound enzyme.”

In one embodiment of the present invention, the capture agent is alectin. Lectins represent a family of saccharide-recognizing proteinswhich are classified based upon the specificity of the mono-saccharidegroups for which they exhibit the highest affinities. Lectins arenon-enzymatic and non-immune in nature and are capable of binding to thesaccharide moiety of a glycoprotein or glycoenzyme. As used herein, theterm “lectin” refers to a non-antibody compound which binds to aspecific carbohydrate structure or target, such as for example, aglycosylated biological or recombinantly derived molecule or aglycosylated enzyme, to form a larger complex. When used in accordancewith the present invention, one or more lectins are selected based uponsuch lectins' affinity for a specific glycan structure or a glycosylatedenzyme. Preferably, the lectin is selected for its biological affinityfor a specific glycan structure, or for a targeted glycosylated enzymeof interest whose presence is suspected in a test sample. In a preferredembodiment the methods and kits of the present invention contemplate theselection of lectins which are capable of recognizing anddiscriminatorily binding to specific glycoforms of an enzyme. Forexample, if the enzyme of interest in a selected test sample is aglycosylated form of the enzyme agalsidase alpha, then a lectin withaffinity for that enzyme (such as, for example, the lectin concanavalinA) would be preferentially incorporated into the assays, kits andmethods of the present invention. By way of further example, if theenzyme of interest in a selected test sample is a glycosylated form ofthe enzyme idursulfase, then a lectin with affinity for that enzyme(such as, for example, SNA Lectin) would be preferentially incorporatedinto the assays, kits and methods of the present invention.

The lectin's biological affinity for a specific glycan structure can beexploited in accordance with the present invention to isolateglycosylated enzymes or specific glycoforms of an enzyme in a testsample. Numerous lectins are commercially available, and information onthe binding specificity of a given lectin can be obtained from themanufacturer or as is described herein. Representative lectins include,but are not limited to, concanavalin A (Con A), wheat germ agglutinin(WGA), Jacalin, lentil lectin (LCA), peanut lectin (PNA), Lens culinarisagglutinin (LCA), Griffonia (Bandeiraea) simplicifolia lectin II (GSLII)Aleuria aurantia Lectin (AAL), Hippeastrum hybrid lectin (HHL, AL),Sambucus nigra lectin (SNA), Maackia amurensis lectin II (MAL II), Ulexeuropaeus agglutinin I (UEA I), Lotus tetragonolobus lectin (LTL),Galanthus nivalis lectin (GNL), Euonymus curopaeus lectin (EEL), Ricinuscommunis agglutinin I (RCA), or combinations thereof.

In an alternative embodiment of the present invention, the capture agentmay comprise one or more fusion proteins, wherein such fusion proteinspreferably comprise one or more binding domains which are capable ofrecognizing and binding to one or more specific saccharide orcarbohydrate moieties of a glycosylated enzyme. For example themannose-6-phosphate receptor (M6PR) is capable of bindingmannose-6-phophate (M6P). The M6PR is approximately 300 kDa and consistsof approximately 15 extracellular domains. In one embodiment of theinvention a fusion protein capture agent comprises one or more M6PRbinding domains (e.g., M6PR domains 1, 3, 5, 9 and/or 11) whichdemonstrate affinity for M6P and/or other glycoforms of interest.Preferably, the selected binding domain demonstrates high affinity forthe saccharide or carbohydrate moieties of interest (e.g., M6PR domains3 and 9 correspond to high affinity M6P binding sites). The recombinantfusion protein capture agents of the present invention may optionallycomprise one or more regions or tags to facilitate purification,isolation or fixation of the capture agent (e.g., fixation to a solidsupport). For example, in one embodiment of the present invention sixhistidine residues (6His) may be linked to a M6PR binding domain tofacilitate the purification, capture or fixation of the capture agent toa solid support (e.g., a nickel chelate-coated 96-well plate). Thefusion protein capture agents of the present invention may furthercomprise one or more linker or spacer groups. In one embodiment of thepresent invention, the capture agent comprises the fusion proteinM6PR(D9)6His which may be expressed, for example, in HT1080 cells andpurified using nickel chelate affinity chromatography.

The present invention contemplates that capture agents (e.g., lectins)may optionally be fixed onto any portion of a solid support (e.g., maybe attached to an interior portion of a porous solid support material).As used herein, the terms “fixed”, “affixed” and “fixing” mean bound,adhered to or immobilized, for example, physically and/or chemically. Asthe term specifically relates to a solid support and a capture agent,“fixed” or “affixed” mean that the capture agent remains bound to, orimmobilized on, a solid support despite being subjected to washconditions or conditions which may alter such physical or chemicalbonds. Fixing may be, for example, spontaneous or result from anadditional step. Exemplary methods of fixing include evaporation (forexample, removal of volatile solvent), cooling (for example, resultingin a phase change from liquid to solid, or viscosity thickening), andcuring (for example, polymerization and/or crosslinking). The presentinvention contemplates the use of derivatized lectins as capture agentsto enhance the ability to fix a lectin onto a solid support. Forexample, biotinylated lectins demonstrate an enhanced ability to affixonto a solid support coated with avidin or streptavidin, (e.g., a 96well plate, optionally coated with avidin or streptavidin) and the useof such derivatized lectins are contemplated by the present invention.(Thompson et al, 1989, Clin. Chim. Acta 180(3):277-84). Other usefulderivatives include, but are not limited to, labels, fluorescent probessuch as rhodamine, or FITC, radioactive labels, electroactive labels,affinity tags (e.g., 6His) that can conjugate with secondary labels,oligonucleotides for PCR amplification, such as green fluorescentprotein or luciferase, chromogenic peptides, and any combinationsthereof.

In one embodiment of the present invention an array of biotinylatedlectins of differing carbohydrate specificities is immobilized directlyonto wells of streptavidin coated 96-well microtiter plates asillustrated in FIG. 1. Test samples containing different preparations ofa given glycosylated enzyme (e.g., aryl sulfatase A (ARSA), agalsidasealfa, galactocerebrosidase (GalC) or heparan N-sulfatase (HNS)) areallowed to bind and unbound material removed by a wash step.

One aspect of the present invention involves the identification andselection of capture agents (e.g., lectins) that demonstrate an affinityfor a glycan structure of interest, or for specific glycoforms of arecombinantly prepared enzyme and a determination that such lectins aresuitable for binding to the specific enzyme glycoform of interest. Theparticular capture agent selected for use in the present invention isgenerally determined based on the ability of such capture agent to bindto a specific glycosylation structure, such as mannose-6-phosphate,fucose, sialic acid, galactose, or any other specific sugar. In somecases, selection of a capture agent is based on the glycosylation of theenzyme targeted for capture. In other cases, the binding agents areselected based on empirical determinations such as high throughputassays. For example, a capture agent can be bound to a solid support andthe ability of the capture agent to bind a desired fraction ofglycosylated enzyme(s) may be determined by using means known to one ofordinary skill in the art (e.g., an ELISA assay). A capture agent foruse in the methods, assays and kits of the present invention may then beselected based upon such capture agent's ability to bind the glycanstructure of interest or a particular glycoform of an enzyme in a testsample. A suitable capture agent for use in the present invention maythen be used to identify the presence or absence of a glycosylatedenzyme of interest in a test sample. For example, such capture agentsmay be used to determine the presence of a particular glycoform of arecombinant enzyme in a harvest test sample extracted from variousstages in the development process of that recombinant enzyme.

A determination of the binding affinity of a particular capture agentfor a glycosylated enzyme of interest may be performed by fixing a panelof labeled capture agents (e.g., biotinylated lectins) on a solidsupport (e.g., a 96 well plate, optionally coated with avidin orstreptavidin). The capture agent panel is then contacted with a testsample suspected of containing the glycosylated enzyme of interest. Inthe presence of the glycosylated enzyme of interest, such enzyme willbind to the capture agent panel and form a bound enzyme complex.

In general, the compositions of the present invention are prepared byattaching (e.g., covalently attaching) at least two (e.g., at leastthree, at least four, at least five or more) capture agents onto a solidsupport or to a molecule that is attached to a solid support (e.g.,avidin, streptavidin or a nickel chelate). One embodiment of the presentinvention contemplates the selection of multiple capture agents whichare prepared by physically mixing at least two (e.g., three, four, five,or more) distinct capture agents and that are subsequently fixed on oneor more solid supports. The amount of a specific capture agent that isselected may be based on the test sample concentration and theapproximate concentration of the target glycoenzyme of interest in thattest sample. In general, the amount of the capture agent fixed onto asolid support to be contacted by a test sample is in excess of at leastabout 50% (e.g., at least 75% or at least 100%) of the amount of theportion of the target glycosylated enzyme predicted to bind to thecapture agent. Alternatively, capture agents are immobilized on a solidsupport at various capture agent/solid support ratios or concentrations.The binding capacity of the capture agent/solid support composition maythen be determined, or the amount of a test sample that can be loadedwithout saturating the solid support may be determined. In general, itis desirable that the amount of capture agent fixed on the solid supportis at least two-fold in excess of the amount of glycosylated enzyme thatis to be bound (e.g., a ten-fold excess or a 100-fold excess).

The invention provides, in part, a capture agent (e.g., multi-captureagent) affinity kit for use in practicing the methods and assays of theinvention. The kits of the present invention may include, for example,at least two capture agents (e.g., lectins) bound to one or more solidsupports. Examples of such solid supports include, without limitation,one or more beads or microbeads composed of silica, agarose, or apolymer, a plate (e.g., a microtiter plate), a slide (e.g., glass orpolymer slide), a nanowell plate, or polyethylene glycol or othersoluble polymer that can be precipitated or isolated by some otherphysical process to which a capture agent is bound. The capture agentsused in the invention can be attached to the solid support directly orindirectly (e.g., using an antibody or biotin) using methods known tothose of ordinary skill in the art, (e.g., using aldehyde functionalizedresins or linkers such as cyanogen bromide, carbonyl diimidazoleglutaraldehyde, epoxy, periodate, or bisoxirane) (Harris et al, 1989, InProtein Purification Methods. A Practical Approach, IRL Press, New York,N.Y.). In the case of particulate supports such as agarose beads, amixture of capture agents (e.g., lectins) may be fixed onto a singlebead, or in certain embodiments, a single type of capture agent isattached to each bead and the mixture of capture agents used in thecomposition is prepared by mixing at least two of these beadpopulations. Alternatively the capture agent may be attached to arestricted access media for the purposes of selecting glycosylatedenzymes of different molecular weight ranges.

The present invention further contemplates the binding of glycosylatedenzymes present in a selected test sample, (e.g., recombinant enzymes)to the capture agent to produce a bound enzyme. As used herein, aglycosylated enzyme is said to have “bound” its respective capture agentwhen it has associated with the capture agent through a non-randomchemical or physical interaction. The terms “bind” or “bound” mean thecoupling of one molecule (e.g., a glycosylated enzyme, such as theenzymes idursulfase, heparan N-sulfatase, arylsulfatase A, agalsidasealfa and galactocerebrosidase) to another molecule (e.g., a lectin, suchas the lectins concanavalin A, sambucus nigra agglutinin and wheat germagglutinin). Binding of an enzyme to a capture agent is preferablyachieved under conditions suitable for such binding to occur, Bindingmay be achieved by chemical means (e.g., covalent or non-covalent innature); however in a preferred embodiment, binding of the capture agentto the glycosylated enzyme of interest in the test sample is achieved byway of a covalent bond. Such binding need not be covalent or permanent.Following contact of the capture agent with the test sample, the testsample is preferably contacted with a wash solution such that the excessor unbound test sample fraction can be separated or removed, and ifappropriate collected for analysis.

The present invention further contemplates detecting the extent to whicha bound enzyme (e.g., the presence of a glycosylated enzyme of interestbound to a capture agent in a selected test sample e.g., recombinantenzymes) competitively inhibits the binding of a ligand to the captureagent bound to the glycosylated enzyme of interest in the test sample.The extent to which a bound enzyme inhibits the binding of a ligand tothe capture agent bound to the glycosylated enzyme correlates to therelative amount of glycan associated with the glycosylated enzyme. Forexample, if the glycosylated enzyme of interest is sialic acidglycosylated idursulfase, and the capture agent is SNA lectin, theextent to which sialic acid glycosylated idursulfase bound to SNA lectininhibits the binding of a ligand (e.g., agalisidase alfa) to the captureagent, thereby inhibiting the dissociation of idursulfase from SNAlectin, correlates to and thus is indicative of the relative amount ofsialic acid content of the glycosylated idursulfase. By way of furtherexample, if the glycosylated enzyme of interest is mannose-6-phosphateglycosylated heparan N-sulfatase, and the capture agent is MP6R, theextent to which mannose-6-phosphate glycosylated heparan N-sulfatasebound to MP6R inhibits the binding of a ligand (e.g., agalsidase alfa)to the capture agent, thereby inhibiting the dissociation of heparanN-sulfatase from MP6R, correlates to and thus is indicative of therelative amount of mannose-6-phosphate content of the glycosylatedheparan N-sulfatase. Thus, the extent to which the glycosylated enzymeinhibits the binding of the ligand to the capture agent of the boundenzyme is indicative of the relative amount of glycan associated withthe glycosylated enzyme present in the test sample. Generally, thegreater the extent to which the bound enzyme inhibits the binding of theligand to the capture agent, the greater the relative amount of glycanassociated with the enzyme in the test sample. Conversely, the lesserthe extent to which the bound enzyme inhibits the binding of the ligandto the capture agent, the lesser the relative amount of glycanassociated with the enzyme in the test sample. It should be appreciatedby those of ordinary skill in the art that any combination of captureagent, ligand, and glycosylated enzyme of interest can be used in thepractice of the assays and methods of the present invention as long asthe ligand and the glycosylated enzyme of interest have been shown tobind to the selected capture agent in a way that correlates to therelative amount of glycan associated with the particular glycosylatedenzyme.

The extent to which the bound enzyme competitively inhibits binding ofthe ligand to the capture agent is generally performed by contacting thebound enzyme with the ligand. It should be appreciated that any ligandwhich is a competitive inhibitor which competes with an enzyme suspectedof being present in a test sample (e.g., cell harvest sample) forbinding to the selected capture agent can be used. In one exampleembodiment, if the enzyme suspected of being present in the test sampleis idursulfase, the competitive inhibitor is agalsidase alfa. In anotherexample embodiment, if the enzyme suspected of being present in the testsample is heparan N-sulfatase, then the competitive inhibitor isagalsidase alfa. In yet another example embodiment, if the enzymesuspected of being present in the test sample is aryl sulfatase A, thecompetitive inhibitor is agalsidase alfa. Following contact of the boundenzyme with the ligand, the test sample may optionally be contacted witha wash solution such that the excess or unbound test sample fraction(e.g., ligand inhibited from binding to capture agent and glycosylatedenzyme dissociated from capture agent) can be separated or removed, andif appropriate collected for analysis.

Generally, the specificity in the detection of the extent to which thebound enzyme inhibits the binding of the ligand to the capture agentwill be performed by determining the amount of ligand bound to thecapture agent. Determining the amount of ligand bound to the captureagent is performed by detecting the intrinsic enzymatic activity of theligand bound to the capture agent via addition of appropriate substrateand assay conditions according to the methods of the present invention.The signal determined for a given capture agent (e.g., a lectin) coupledwith the known specificity of that capture agent will allow for a fast,high throughput, semi-quantitative structure assessment of theglycoforms present in the test sample. To determine the relative amountof glycan associated with a glycosylated enzyme of interest in a testsample, the bound enzyme fraction (e.g., including both the glycosylatedenzyme bound to capture agent and the ligand bound to capture agent) iscontacted with a substrate. As used herein, the term “contact” meansthat two or more substances (e.g., a bound enzyme and a substrate orligand bound to capture agent and a substrate) are sufficiently close toeach other such that the two or more substances interact or react (e.g.,chemically or biologically) with one another. The term “substrate”refers to a molecule, complex, material, substance or reactant withwhich an enzyme reacts (e.g., chemically or biologically), acts orbinds. In particular, the substrates of the present invention maydemonstrate a physiological, biological and/or chemical affinity for, orbe able to be acted upon, by a corresponding enzyme. A substrate usefulin the methods of the invention can be native or modified. Modifiedsubstrates useful in the invention retain the ability to be acted uponby the corresponding enzyme. Exemplary modifications suitable forsubstrates include, for example, labeling to confirm the presence orabsence of intrinsic enzymatic activity. One aspect of the presentinvention contemplates the selection of substrates based upon itsability to interact with, or bind to the ligand in a predictable andrepeatable fashion. For example, a substrate with which a ligand (e.g.,enzyme) is known to react would be preferable. Once a suitable substratehas been identified, that substrate is preferably contacted with thebound enzyme and the ligand bound to the capture agent, and the presenceor absence of the anticipated reaction or interaction is assessed.

Based upon the known intrinsic enzymatic activity of the ligand, in thepresence of the appropriate substrate the ligand would be expected toreact, and accordingly signal the amount of that ligand in the testsample. For example, the product of an enzyme reaction (e.g., a ligand,such as agalsidase alfa, for example) with a substrate (e.g., amolecular entity that is produced or liberated as a result of enzymeacting on substrate) may provide a measurable signal indicative of thepresence of ligand in the test sample, and that correlates with thepresence or amount of intrinsic enzymatic activity of the ligand in thetest sample. Alternatively, quantitative assessments of substratebinding and/or assessments of the conversion of substrate to product maybe performed and used as a surrogate marker of intrinsic enzymaticactivity of the ligand.

Generally, a greater amount of intrinsic enzymatic activity of theligand detected is indicative of a lesser extent to which the boundenzyme inhibits the binding of the ligand to the capture agent, and alesser amount of intrinsic enzymatic activity detected is indicative ofa greater extent to which the bound enzyme inhibits the binding of theligand to the capture agent. Said differently, a greater amount ofintrinsic enzymatic activity of the ligand bound to the capture agentdetected is indicative of a lesser relative amount of glycan associatedwith the glycosylated enzyme of interest in the test sample, and alesser amount of intrinsic enzymatic activity of the ligand bound to thecapture agent is indicative of a greater relative amount of glycanassociated with the glycosylated enzyme of interest in the test sample.

Examples of suitable substrates for use in the present invention include4-nitrophenyl-α-D-galactopyranoside, 4-nitrophenyl-β-D-galactopyranosideand para-nitrocatechol sulfate.

Selection of the appropriate enzyme (e.g., ligand) substrate and thesubsequent determination of intrinsic enzymatic activity of the enzymerequire an understanding of enzyme kinetics and in particular thecatalytic properties of the enzyme(s) being evaluated. For example,enzymatic properties, such as Michaelis-Menton constants (K_(m)) and/orturnover numbers (K_(cat)) relating to a particular substrate providethe basis for evaluating the sensitivity of an enzyme for one or moresubstrates and provide information regarding the reproducibility of themethods, kits and assays contemplated by the present inventions.

As used herein, the term “intrinsic enzymatic activity” refers to therepeatable reaction which an enzyme (e.g., ligand) catalyzes or causesto occur, for example in the presence of a substrate with which suchenzyme is known to react. In one embodiment of the present invention,the intrinsic enzymatic activity of ligand may be exploited to confirmthe presence or absence of such ligand in a particular test sample. Forexample, many enzymes have known and repeatable catalytic activity,which may be enhanced under certain conditions, such as the presence ofthe appropriate substrate. Intrinsic enzymatic activity may be measuredby routine means known to one of ordinary skill in the art (e.g.,colorimetric, spectrophotometric, fluorometric or chromatographicassays) by determining, for example the production of a product overtime. In accordance with the present invention, following contacting aligand with a substrate, a relative increase in the formation of aproduct, or the conversion of that substrate to product, in each case ofabout 5%, 10%, 20%, 30%, 40%, 50% or more, or preferably about 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more preferably 100% or more,may be indicative of intrinsic enzymatic activity of the ligand.

The methods described herein are useful for development of kits that canbe used for the detection of enzymes and the relative amount of glycanassociated with the enzymes in a test sample. Such kits include one ormore capture agents (e.g., lectins or fusion proteins) fixed on a solidsupport which are capable of binding to a glycosylated enzyme present ina selected test sample. The kits may also include additional reagentssuch as buffers, substrates, enzymes, chemicals and other compositionsuseful for further analysis and/or quantification of the ligand-boundcapture agent fraction. Kits can also include components for test samplepreparation. The methods and kits described herein are useful forproviding a platform for the semi-quantitative assessment of thepresence of glycosylated enzymes and the relative amount of glycanassociated with the glycosylated enzyme in a test sample.

While certain compounds, compositions and methods of the presentinvention have been described with specificity in accordance withcertain embodiments, the following examples serve only to illustrate themethods, assays, kits and compositions of the invention and are notintended to limit the same.

The articles “a” and “an” as used herein in the specification and in theclaims, unless clearly indicated to the contrary, should be understoodto include the plural referents. Claims or descriptions that include“or” between one or more members of a group are considered satisfied ifone, more than one, or all of the group members are present in, employedin, or otherwise relevant to a given product or process unless indicatedto the contrary or otherwise evident from the context. The inventionincludes embodiments in which exactly one member of the group is presentin, employed in, or otherwise relevant to a given product or process.The invention also includes embodiments in which more than one, or theentire group members are present in, employed in, or otherwise relevantto a given product or process. Furthermore, it is to be understood thatthe invention encompasses all variations, combinations, and permutationsin which one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the listed claims is introduced into anotherclaim dependent on the same base claim (or, as relevant, any otherclaim) unless otherwise indicated or unless it would be evident to oneof ordinary skill in the art that a contradiction or inconsistency wouldarise. Where elements are presented as lists, (e.g., in Markush group orsimilar format) it is to be understood that each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should be understood that, in general, where the invention, oraspects of the invention, is/are referred to as comprising particularelements, features, etc., certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements, features, etc. For purposes of simplicity those embodimentshave not in every case been specifically set forth in so many wordsherein. It should also be understood that any embodiment or aspect ofthe invention can be explicitly excluded from the claims, regardless ofwhether the specific exclusion is recited in the specification. Thepublications and other reference materials referenced herein to describethe background of the invention and to provide additional detailregarding its practice are hereby incorporated by reference.

Example 1

Studies performed using the inventions disclosed herein havedemonstrated carbohydrate-specific binding of agalsidase alfa andgalactocerebrosidase drug substance material to several capture agents.The capture agents evaluated included the biotinylated lectins ConA(specific for core α-mannose structures), WGA (specific for dimers andtrimers of N-acetyl-glucosamine), and SNA (specific for Neu5Ac(α2,6)Galstructures).

Technical Feasibility

To determine the technical feasibility of the methods, assays andcompositions described herein, purified agalsidase alfa, aryl sulfataseA and galactocerebrosidase samples were initially assessed. The choiceof capture agent was initially limited to only a select few lectins withwell defined binding specificities. The lectin capture agents utilizedincluded: (1) concanavalin A (Con A), one of the most commonly usedlectins and known to bind avidly to core α-mannose structures ofN-linked high-mannose and biantennary complex-type oligosaccharides, (2)Sambucus nigra lectin (SNA) and Maackia amurensis lectin (MAL), whichrecognize Neu5Ac(a2,6)Gal and Neu5Ac(a2,3)Gal respectively, (3) Ricinuscommunis agglutinin I (RCAI), which binds terminal β1,4-linked Gal, and(4) wheat germ agglutinin (WGA), which binds poly-lactosamine repeatsGalβ1,4GlcNAc. Technical feasibility was based on sensitivity ofdetection. Feasibility was further evaluated using in-process testsamples provided from cell culture/process development streams.

Results

Method feasibility was evaluated using agalsidase alfa (Replagal®) drugsubstance and the biotinylated lectins ConA and WGA as the captureagents. The binding of decreasing agalsidase alfa concentrations (40ug/mL to 300 ng/mL) to the immobilized lectins was analyzed by measuringthe enzymatic activity of the bound enzyme. The enzyme activity ofagalsidase alfa was determined under steady-state conditions for thesynthetic colorimetric substrate 4-nitrophenyl-α-D-galactopyranoside.The substrate was hydrolyzed into 4-nitrophenol andα-D-galactopyranoside where the p-nitrophenol product was read at 405 nmonce the reaction was halted with an alkaline buffer. The binding curvesof agalsidase alfa (Replagal®) by both WGA and ConA (in absorbanceunits) are shown in FIG. 2.

The high avidity for ConA (which is specific for α-linked mannosestructures) at all concentrations tested demonstrated the highsensitivity of the assays and methods described herein. The binding forWGA (which is specific for dimers and trimers of N-acetyl-glucosamine)was less avid and followed a more classical titration curve. Thesestudies demonstrated the potential of the methods and assays of thepresent invention in terms of their sensitivity and high-throughputnature.

Example 2

Method feasibility was further evaluated with galactocerebrosidase(GalC) drug substance material and the biotinylated lectins concanavalinA (Con A), wheat germ agglutinin (WGA), and Sambucus nigra lectin (SNA).The binding curves in FIG. 3 demonstrate both strong and selectivebinding to GalC.

To determine whether the methods and assays of the present inventionwere capable of detecting differences in the amount of GalC-associatedsialic acid, GalC samples were subjected to increasing amounts ofsialidase pre-treatment and then evaluated for binding. The resultsprovided in FIG. 4 demonstrate that controlled removal of sialic acidresults in reduced binding, providing evidence that the assays andmethods of the present invention are capable of measuring differences inthe amount of GalC-associated sialic acid.

Example 3

To confirm the sialic acid binding specificity of Sambucus nigra lectin(SNA), aryl sulfatase A drug substance samples produced in CHO and humancells containing approximately 1 mol of sialic acid per mol of proteinin either α2,6-linkage (human cell-derived) or α2,3-linkage (CHOcell-derived) were analyzed for binding.

The binding curves provided in FIG. 5 illustrate both the selectivebinding for sialic acid in the α2,6-linkage and the α2,3-linkage.

Example 4

To determine whether the assays, methods and compositions describedherein could be applied to actual harvest samples, early, middle, andlate galactocerebrosidase (GalC) harvest samples (H2, H10, and H17) froman early stage in the development process were analyzed for binding toSambucus nigra lectin (SNA).

The results shown in FIG. 6 demonstrate no difference in sialic acidbinding across all 3 harvests and importantly validate the intendedpurpose of the present invention.

Example 5

The feasibility of the present invention was also evaluated with arylsulfatase A drug substance material derived from two differentproduction methods and a recombinant fusion protein which was preparedto function as the capture agent. The recombinant fusion proteinconsisted of the high affinity binding domain (D9) of themannose-6-phosphate receptor (M6PR) linked to six histidine residues(6His) to facilitate fixation of the M6PR to a nickel chelate coatedsolid support. The recombinant fusion protein construct is referred toherein as M6PR(D9)6His and was expressed in HT1080 cells, purified usingnickel chelate affinity chromatography, and was affixed onto a 96-wellplate.

To determine whether the methods and assays of the present inventionwere capable of detecting differences in the amount of aryl sulfatase Aassociated M6P associated with the two different production methods,aryl sulfatase A lots with known amounts of M6P were evaluated forbinding to immobilized M6PR(D9)6His (FIG. 7). Increasing concentrationsof test samples designated as either HGT-1110 or HGT-1111 (correspondingto the respective production methods) were added to the wells andallowed to bind for 2 hours at room temperature. The wells were washedin PBS and the enzyme activity was measured using the substratep-nitrocatechol sulfate.

As shown in FIG. 8, aryl sulfatase A from lot HGT-1111 which known toinclude more that 2×M6P per mol of protein as compared to aryl sulfataseA from HGT-1111, displayed more avid binding as compared to arylsulfatase A from lot HGT-1110. These results demonstrate that themethods, assays and kits of the present invention are capable ofmeasuring differences in the amount of aryl sulfatase A-associated M6P.

Example 6

Agalsidase alfa has been demonstrated to bind to Sambucus nigra lectin(SNA) in a manner that correlates to the amount of sialic acidassociated with agalsidase alfa. The feasibility of the presentinvention was also evaluated with idursulfase, heparan N-sulfatase(FINS), and aryl sulfatase A (ARSA) drug substance materials ascompetitive inhibitors of the binding of the ligand agalsidase alfa tothe capture agent Sambucus nigra lectin.

To determine whether the methods and assays of the present inventionwere capable of detecting differences in the relative amounts of sialicacid associated with idursulfase, heparan N-sulfatase, and arylsulfatase, agalsidase alfa lots with known amounts of sialic acid wereevaluated for binding to SNA immobilized in 96-well plates in thepresence of increasing concentrations of idursulfase (o), HNS (o), orARSA (A) (FIG. 9). The amount of agalsidase alfa bound, and the halfmaximal inhibitory concentrations (IC₅₀) of each drug substance, wasdetermined by measuring the intrinsic enzymatic activity of agalsidasealfa bound to SNA in accordance with the methods of the presentinvention.

As shown in Table 1 below, idursulfase was the most potent inhibitor ofsialic acid binding with an IC₅₀ of 2.3×10⁻⁸ M, followed by FINS with anIC₅₀ of 2.2×10⁻⁷ M and then ARSA with IC₅₀ 1.2×10⁻⁶ M. Note that theseresults are consistent with the relative amounts of sialic acidassociated with idursulfase, FINS, and ARSA, respectively. These resultsdemonstrate that the methods, assays and kits of the present inventionare capable of detecting the relative amounts of glycan (e.g., sialicacid) associated with a glycosylated enzyme (e.g., such as idursulfase,FINS, and ARSA, for example) in a way that correlates to the extent towhich the glycosylated enzyme competitively inhibits the binding of aligand (e.g., agalsidase alfa) to a capture agent (e.g., SNA lectin).

TABLE 1 Half maximal inhibitory concentrations (IC₅₀) of glycosylatedenzyme inhibiting binding of agalisidase alfa to SNA lectin GlycosylatedEnzyme IC₅₀ (nM) Idursulfase 23 HNS 215 ARSA 1209

Example 7

Agalsidase alfa has been demonstrated to bind to MP6 Receptor (MP6R) ina manner that correlates to the amount of mannose-6-phosphate associatedwith agalsidase alfa. The feasibility of the present invention was alsoevaluated with idursulfase, heparan N-sulfatase (FINS), and arylsulfatase A (ARSA) drug substance materials as competitive inhibitors ofthe binding of the ligand agalsidase alfa to the capture agent MP6R.

To determine whether the methods and assays of the present inventionwere capable of detecting differences in the relative amounts ofmannose-6-phosphate associated with idursulfase, heparan N-sulfatase,and aryl sulfatase, agalsidase alfa lots with known amounts ofmannose-6-phosphate were evaluated for binding to MP6R immobilized in96-well plates in the presence of increasing concentrations ofidursulfase (◯), HNS (□), or ARSA (▴) (FIG. 10). The amount ofagalsidase alfa bound, and the half maximal inhibitory concentrations(IC₅₀) of each drug substance, was determined by measuring the intrinsicenzymatic activity of agalsidase alfa bound to MP6R in accordance withthe methods of the present invention.

As shown in Table 2 below, idursulfase was the most potent inhibitor ofmannose-6-phosphate binding with an IC₅₀ of 1.3×10⁻⁸ M, followed by HNSwith an IC₅₀ of 3.6×10⁻⁸ M and then ARSA with an IC₅₀ 1.9×10⁻⁷ M. Notethat these results are consistent with the relative amounts ofmannose-6-phosphate associated with idursulfase, HNS, and ARSA,respectively. These results demonstrate that the methods, assays andkits of the present invention are capable of detecting the relativeamounts of glycan (e.g., mannose-6-phosphate) associated with aglycosylated enzyme (e.g., such as idursulfase, HNS, and ARSA, forexample) in a way that correlates to the extent to which theglycosylated enzyme competitively inhibits the binding of a ligand(e.g., agalsidase alfa) to a capture agent (e.g., MP6R).

TABLE 2. Half maximal inhibitory concentrations (IC₅₀) of glycosylatedenzyme inhibiting binding of agalisidase alfa to MP6R GlycosylatedEnzyme IC₅₀ (nM) Idursulfase 13 HNS 36 ARSA 189

1-82. (canceled)
 83. A method of detecting a relative amount of glycanassociated with an enzyme in a test sample, the method comprising: (a)contacting the test sample with at least one capture agent underconditions appropriate for binding of glycosylated enzyme in the testsample to the capture agent, wherein if the glycosylated enzyme ispresent in the test sample a bound enzyme is formed; (b) separating thebound enzyme from the test sample; and (c) detecting the extent to whichthe bound enzyme inhibits binding of a ligand to the capture agent,wherein the extent to which the bound enzyme inhibits binding of theligand to the capture agent is indicative of the relative amount ofglycan associated with the enzyme in the test sample; wherein thecapture agent comprises a biotinylated lectin.
 84. The method of claim83, wherein the lectin is concanavalin A, wheat germ agglutinin,Jacalin, lentil lectin, peanut lectin, lens culinaris agglutinin,Griffonia (Bandeiraea) simplicifolia lectin II, Aleuria aurantia lectin,hippeastrum hybrid lectin, sambucus nigra lectin, maackia amurensislectin II, ulex europaeus agglutinin I, lotus tetragonolobus lectin,galanthus nivalis lectin, euonymus europaeus lectin, or ricinus communisagglutinin I.
 85. The method of claim 83, wherein the lectin is affixedon a solid support.
 86. The method of claim 85, wherein the solidsupport comprises avidin or streptavidin.
 87. The method of claim 85,wherein the solid support comprises a multiple well microtiter plate.88. The method of claim 86, wherein the solid support comprises at leastone bead.
 89. The method of claim 83, wherein the step of detecting theextent to which the bound enzyme inhibits binding of the ligand to thecapture agent is performed by contacting the bound enzyme with theligand.
 90. The method of claim 89, wherein the ligand is a competitiveinhibitor which competes with an enzyme suspected of being present inthe test sample for binding to the capture agent.
 91. The method ofclaim 90, wherein the enzyme is idursulfase and the competitiveinhibitor is agalsidase alfa.
 92. The method of claim 90, wherein theenzyme is heparan N-sulfatase and the competitive inhibitor isagalsidase alfa.
 93. The method of claim 90, wherein the enzyme is arylsulfatase A and the competitive inhibitor is agalsidase alfa.
 94. Themethod of claim 83, wherein the greater the extent to which the boundenzyme inhibits binding of the ligand to the capture agent is indicativeof a greater relative amount of glycan associated with the enzyme in thetest sample, and wherein the lesser the extent to which the bound enzymeinhibits binding of the ligand to the capture agent is indicative of alesser relative amount of glycan associated with the enzyme in the testsample.
 95. The method of claim 89, wherein the step of detecting theextent to which the bound enzyme inhibits binding of the ligand to thecapture agent comprises determining the amount of the ligand bound tothe capture agent.
 96. The method of claim 95, wherein the step ofdetermining the amount of the ligand bound to the capture agentcomprises detecting the intrinsic enzymatic activity of the ligand boundto the capture agent.
 97. The method of claim 96, wherein the step ofdetecting intrinsic enzymatic activity of the ligand bound to thecapture agent is performed by contacting the ligand bound to the captureagent with a substrate.
 98. The method of claim 97, wherein thesubstrate is reactive with the ligand bound to the capture agent. 99.The method of claim 97, wherein the conversion of the substrate toproduct is indicative of intrinsic enzymatic activity.
 100. The methodof claim 96, wherein the step of detecting intrinsic enzymatic activitycomprises quantitatively determining the presence of a product formedafter contacting the ligand bound to the capture agent with thesubstrate.
 101. The method of claim 100, wherein the presence of theproduct is indicative of intrinsic enzymatic activity.
 102. The methodof claim 97, wherein the ligand is agalsidase alfa and the substrate is4-nitrophenyl-α-D-galactopyranoside.
 103. The method of claim 83,wherein the enzyme is idursulfase and the glycan is sialic acid. 104.The method of claim 83, wherein the enzyme is idursulfase and the glycanis mannose-6-phosphate.
 105. The method of claim 83, wherein the enzymeis heparan N-sulfatase and the glycan is sialic acid.
 106. The method ofclaim 83, wherein the enzyme is heparan N-sulfatase and the glycan ismannose-6-phosphate.
 107. The method of claim 83, wherein the enzyme isaryl sulfatase A and the glycan is sialic acid.
 108. The method of claim83, wherein the enzyme is aryl sulfatase A and the glycan ismannose-6-phosphate.
 109. The method of claim 83, wherein the step ofseparating the bound enzyme from the test sample is performed bywashing.
 110. A method of detecting a relative amount of glycanassociated with an enzyme in a test sample, the method comprising: (a)contacting a test sample with at least one capture agent underconditions appropriate for binding of glycosylated enzyme, wherein ifthe glycosylated enzyme is present in the test sample a bound enzyme isformed; (b) separating the bound enzyme from the test sample; (c)contacting the bound enzyme with at least one ligand for the captureagent; and (d) detecting the extent to which the bound enzyme inhibitsbinding of the ligand to the capture agent, wherein the extent to whichthe bound enzyme inhibits binding of the ligand to the capture agent isindicative of the relative amount of glycan associated with the enzymein the test sample; wherein the capture agent comprises a biotinylatedlectin.
 111. The method of claim 110, wherein the lectin is concanavalinA, wheat germ agglutinin, Jacalin, lentil lectin, peanut lectin, lensculinaris agglutinin, Griffonia (Bandeiraea) simplicifolia lectin II,Aleuria aurantia lectin, hippeastrum hybrid lectin, sambucus nigralectin, maackia amurensis lectin II, ulex europaeus agglutinin I, lotustetragonolobus lectin, galanthus nivalis lectin, euonymus europaeuslectin, or ricinus communis agglutinin I.
 112. The method of claim 110,wherein the lectin is affixed on a solid support.
 113. The method ofclaim 112, wherein the solid support comprises avidin or streptavidin.114. The method of claim 112, wherein the solid support comprises amultiple well microtiter plate.
 115. The method of claim 113, whereinthe solid support comprises at least one bead.
 116. The method of claim110, wherein the ligand is a competitive inhibitor which competes withan enzyme suspected of being present in the test sample for binding tothe capture agent.
 117. The method of claim 116, wherein the enzyme isidursulfase and the competitive inhibitor is agalsidase alfa.
 118. Themethod of claim 116, wherein the enzyme is heparan N-sulfatase and thecompetitive inhibitor is agalsidase alfa.
 119. The method of claim 116,wherein the enzyme is aryl sulfatase A and the competitive inhibitor isagalsidase alfa.
 120. The method of claim 110, wherein the greater theextent to which the bound enzyme inhibits binding of the ligand to thecapture agent is indicative of a greater relative amount of the glycanassociated with the enzyme glycoform in the test sample, and wherein thelesser the extent to which the bound enzyme inhibits binding of theligand to the capture agent is indicative of a lesser relative amount ofglycan associated with the enzyme glycoform in the test sample.
 121. Themethod of claim 110, wherein the step of detecting the extent to whichthe bound enzyme inhibits binding of the ligand to the capture agentcomprises determining the amount of the ligand bound to the captureagent.
 122. The method of claim 121, wherein the step of determining theamount of the ligand bound to the capture agent comprises detecting theintrinsic enzymatic activity of the ligand bound to the capture agent.123. The method of claim 122, wherein the step of detecting intrinsicenzymatic activity of the ligand bound to the capture agent is performedby contacting the ligand bound to the capture agent with a substrate.124. The method of claim 123, wherein the substrate is reactive with theligand bound to the capture agent.
 125. The method of claim 123, whereinthe conversion of the substrate to product is indicative of intrinsicenzymatic activity.
 126. The method of claim 122, wherein the step ofdetecting intrinsic enzymatic activity comprises quantitativelydetermining the presence of a product formed after contacting the ligandbound to the capture agent with the substrate.
 127. The method of claim126, wherein the presence of the product is indicative of intrinsicenzymatic activity.
 128. The method of claim 123, wherein the ligand isagalsidase alfa and the substrate is4-nitrophenyl-α-D-galactopyranoside.
 129. The method of claim 122,wherein the enzyme is idursulfase and the glycan is sialic acid. 130.The method of claim 122, wherein the enzyme is idursulfase and theglycan is mannose-6-phosphate.
 131. The method of claim 122, wherein theenzyme is heparan N-sulfatase and the glycan is sialic acid.
 132. Themethod of claim 122, wherein the enzyme is heparan N-sulfatase and theglycan is mannose-6-phosphate.
 133. The method of claim 122, wherein theenzyme is aryl sulfatase A and the glycan is sialic acid.
 134. Themethod of claim 122, wherein the enzyme is aryl sulfatase A and theglycan is mannose-6-phosphate.
 135. The method of claim 122, wherein thebound enzyme is separated from the test sample by washing.
 136. A kitfor detecting the relative amount of glycan associated with anglycosylated enzyme in a test sample, wherein the kit comprises (a) atleast one capture agent, wherein the capture agent is capable of bindingthe glycosylated enzyme, and (b) at least one ligand, wherein the ligandis competitive with the glycosylated enzyme for binding the at least onecapture agent; wherein the capture agent comprises a biotinylatedlectin.
 137. The kit of claim 136, wherein the lectin is concanavalin A,wheat germ agglutinin, Jacalin, lentil lectin, peanut lectin, lensculinaris agglutinin, Griffonia (Bandeiraea) simplicifolia lectin II,Aleuria aurantia lectin, hippeastrum hybrid lectin, sambucus nigralectin, maackia amurensis lectin II, ulex europaeus agglutinin I, lotustetragonolobus lectin, galanthus nivalis lectin, euonymus europaeuslectin, or ricinus communis agglutinin I.
 138. The kit of claim 136,wherein the kit further comprises a solid support.
 139. The kit of claim138, wherein the lectin is affixed on the solid support.
 140. The kit ofclaim 138, wherein the solid support comprises avidin or streptavidin.141. The kit of claim 138, wherein the solid support comprises amultiple well microtiter plate.
 142. The kit of claim 140, wherein thesolid support comprises at least one bead.
 143. The kit of claim 136,wherein the glycosylated enzyme is selected from the group consisting ofidursulfase, heparan N-sulfatase, and aryl sulfatase A.
 144. The kit ofclaim 136, wherein the ligand is agalsidase alfa.
 145. The kit of claim136, wherein the ligand is agalsidase alfa, and the glycosylated enzymeis selected from the group consisting of idursulfase, heparanN-sulfatase, and aryl sulfatase A.
 146. The kit of claim 136, furthercomprising a substrate.
 147. The kit of claim 146, wherein the substrateis 4-nitrophenyl-β-D-galactopyranoside.